Since calcium is important in many biological functions and is related to many diseases and health related disorders, the effects of charge distribution, conformation, and metal selectivity to calcium-binding proteins are important for understanding calcium-induced conformational change and binding affinity. This will allow future design of calcium triggers to control protein function and contribute to the understanding of diseases and health related disorders involved in calcium-binding. To initially dissect the role of charge and ligand type to conformation and metal selectivity, calcium-binding proteins (using glutamate as the bidentate ligand) have been designed, expressed, and purified in the host protein, domain 1 of CD2 (CD2-D1). These proteins have different net negative charges in the coordination sphere and different ligand types. Currently, conformational analysis and metal binding affinity and selectivity are being studied using far UV CD, Trp fluorescence, and FRET. Further analysis will be completed using NMR, ICP-MS, near UV CD and ITC. In addition, a new generation of calcium-binding proteins (using aspartate as the bidentate ligand) is being designed and engineered. Our desire for the new generation will be to understand charge induced conformational change and local charge effects to metal binding affinity and selectivity.This will allow us to test the key factors for calcium binding with site-specific metal binding properties. The knowledge obtained will equip us with the ability to generate therapeutic agents for diseases and health disorders related to the improper use of calcium. The studies already completed are encouraging and exciting. They bring us closer to our hypothesis and to our long-term goals.